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- PDB-7usd: Cryo-EM structure of D-site Rac1-bound WAVE Regulatory Complex -

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Basic information

Entry
Database: PDB / ID: 7usd
TitleCryo-EM structure of D-site Rac1-bound WAVE Regulatory Complex
Components
  • Abl interactor 2
  • Cytoplasmic FMR1-interacting protein 1
  • Nck-associated protein 1
  • Protein BRICK1
  • Ras-related C3 botulinum toxin substrate 1
  • Wiskott-Aldrich syndrome protein family member 1
KeywordsCELL INVASION / actin regulator / GTPase binding protein / cytoskeletal regulator
Function / homology
Function and homology information


peripheral region of growth cone / SCAR complex / negative regulation of synaptic vesicle recycling / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / regulation of actin polymerization or depolymerization / central region of growth cone / modification of postsynaptic actin cytoskeleton ...peripheral region of growth cone / SCAR complex / negative regulation of synaptic vesicle recycling / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / regulation of actin polymerization or depolymerization / central region of growth cone / modification of postsynaptic actin cytoskeleton / regulation of respiratory burst / dendrite extension / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / regulation of translation at postsynapse, modulating synaptic transmission / Activated NTRK2 signals through CDK5 / regulation of hydrogen peroxide metabolic process / negative regulation of receptor-mediated endocytosis / ruffle assembly / NTRK2 activates RAC1 / filopodium tip / NADPH oxidase complex / Inactivation of CDC42 and RAC1 / regulation of actin filament polymerization / respiratory burst / WNT5:FZD7-mediated leishmania damping / cortical cytoskeleton organization / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / regulation of modification of postsynaptic actin cytoskeleton / hepatocyte growth factor receptor signaling pathway / RNA 7-methylguanosine cap binding / ruffle organization / cell projection assembly / positive regulation of bicellular tight junction assembly / thioesterase binding / regulation of stress fiber assembly / regulation of lamellipodium assembly / negative regulation of fibroblast migration / RHO GTPases activate CIT / regulation of nitric oxide biosynthetic process / motor neuron axon guidance / Nef and signal transduction / sphingosine-1-phosphate receptor signaling pathway / positive regulation of ruffle assembly / axon extension / PCP/CE pathway / Activation of RAC1 / RHO GTPases activate KTN1 / MET activates RAP1 and RAC1 / DCC mediated attractive signaling / Azathioprine ADME / positive regulation of cell-substrate adhesion / positive regulation of neutrophil chemotaxis / Sema4D mediated inhibition of cell attachment and migration / Ephrin signaling / CD28 dependent Vav1 pathway / cortical actin cytoskeleton organization / superoxide anion generation / Wnt signaling pathway, planar cell polarity pathway / lamellipodium assembly / positive regulation of dendrite development / regulation of myelination / positive regulation of actin filament polymerization / Activation of RAC1 downstream of NMDARs / protein kinase A binding / small GTPase-mediated signal transduction / NRAGE signals death through JNK / regulation of cell size / positive regulation of Rho protein signal transduction / Rho GDP-dissociation inhibitor binding / establishment or maintenance of cell polarity / protein kinase A regulatory subunit binding / Rac protein signal transduction / filamentous actin / RHO GTPases activate PAKs / dendritic growth cone / semaphorin-plexin signaling pathway / lamellipodium membrane / ficolin-1-rich granule membrane / excitatory synapse / Sema3A PAK dependent Axon repulsion / RHOG GTPase cycle / RHO GTPases Activate NADPH Oxidases / EPH-ephrin mediated repulsion of cells / positive regulation of focal adhesion assembly / anatomical structure morphogenesis / RAC2 GTPase cycle / RHO GTPases Activate WASPs and WAVEs / RAC3 GTPase cycle / positive regulation of axon extension / RHO GTPases activate IQGAPs / axonal growth cone / response to electrical stimulus / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of lamellipodium assembly / RHO GTPases activate PKNs / GPVI-mediated activation cascade / positive regulation of stress fiber assembly / ruffle
Similarity search - Function
Cytoplasmic FMR1-interacting / Nck-associated protein 1 / Cytoplasmic Fragile-X interacting family / Nck-associated protein 1 / Protein BRICK1 / Abl interactor 2, SH3 domain / CYRIA/CYRIB, Rac1 binding domain / Abl-interactor, homeo-domain homologous domain / SCAR/WAVE family / ABI family ...Cytoplasmic FMR1-interacting / Nck-associated protein 1 / Cytoplasmic Fragile-X interacting family / Nck-associated protein 1 / Protein BRICK1 / Abl interactor 2, SH3 domain / CYRIA/CYRIB, Rac1 binding domain / Abl-interactor, homeo-domain homologous domain / SCAR/WAVE family / ABI family / CYRIA/CYRIB Rac1 binding domain / Abl-interactor HHR / Wiskott Aldrich syndrome homology region 2 / WH2 motif / WH2 domain / WH2 domain profile. / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / Small GTPase Rho / small GTPase Rho family profile. / SH3 domain / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Abl interactor 2 / Ras-related C3 botulinum toxin substrate 1 / Cytoplasmic FMR1-interacting protein 1 / Protein BRICK1 / Actin-binding protein WASF1 / Nck-associated protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsDing, B. / Yang, S. / Chen, B. / Chowdhury, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2022
Title: Structures reveal a key mechanism of WAVE regulatory complex activation by Rac1 GTPase.
Authors: Bojian Ding / Sheng Yang / Matthias Schaks / Yijun Liu / Abbigale J Brown / Klemens Rottner / Saikat Chowdhury / Baoyu Chen /
Abstract: The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the ...The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ~3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2-central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.
History
DepositionApr 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytoplasmic FMR1-interacting protein 1
B: Nck-associated protein 1
C: Wiskott-Aldrich syndrome protein family member 1
D: Protein BRICK1
E: Abl interactor 2
F: Ras-related C3 botulinum toxin substrate 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)359,6708
Polymers359,1236
Non-polymers5472
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 6 types, 6 molecules ABCDEF

#1: Protein Cytoplasmic FMR1-interacting protein 1 / Specifically Rac1-associated protein 1 / Sra-1 / p140sra-1


Mass: 145363.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: CYFIP1, KIAA0068 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q7L576
#2: Protein Nck-associated protein 1 / NAP 1 / Membrane-associated protein HEM-2 / p125Nap1


Mass: 128940.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: NCKAP1, HEM2, KIAA0587, NAP1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Y2A7
#3: Protein Wiskott-Aldrich syndrome protein family member 1 / WASP family protein member 1 / Protein WAVE-1 / Verprolin homology domain-containing protein 1


Mass: 37009.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification ...Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: WASF1, KIAA0269, SCAR1, WAVE1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q92558
#4: Protein Protein BRICK1 / BRK1


Mass: 8756.915 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were ...Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: BRK1, C3orf10, HSPC300, MDS027 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8WUW1
#5: Protein Abl interactor 2


Mass: 18041.482 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these ...Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: ABI2 / Production host: Escherichia coli (E. coli) / References: UniProt: E9PEZ7
#6: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 21010.486 Da / Num. of mol.: 1 / Mutation: P29S, Q61L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63000, small monomeric GTPase

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: WAVE regulatory complex with Rac1 bound to D-site / Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.36 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
31Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenConc.: 0.41 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: Data were collected by shifting the stage to target exposure positions.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 1000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 45.27 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2512
Details: Each micrograph was acquired as dose-fractionated movies consisting of 62 frames per movie.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPU2.1image acquisition
4GctfCTF correction
7NAMDmodel fitting
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
12RELION3.1 betafinal Euler assignment
13RELION3.1 betaclassification
14RELION3.1 beta3D reconstruction
CTF correctionDetails: Particles were CTF-corrected during projection matching and back projection
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1765193
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87810 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13P8C

3p8c

13P8C1PDBexperimental model
23SBD

3sbd

13SBD2PDBexperimental model

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